Wheat (Triticum aestivum L.) microsatellite markers were screened for detecting Haynaldia villosa L. chromosomes introduced into wheat background. Two hundred and seventy six primer pairs mapped on 7 homeologous groups of wheat were used to amplify the gDNA of T. aestivum and H. villosa. The results showed that 148 of 276 microsatellite primers amplified polymorphic bands between common wheat cv. Chinese Spring and H. villosa. Primers wmc49 (1BS), wmc25 (2BS), gdm36 (3DS), gdm145 (4AL), wmc233 (5DS), wmc256 (6AL) and gwm344 (7BL) produced a specific polymorphic DNA fragment on chromosome 1V to 7V of H. villosa, respectively. In addition, gwm469 (6DS) detected a specific band on 2V; gdm107 (2DS) amplified a specific band on 6V. These microsatellite markers were effective in identifying individual H. villosa chromosomes in other T. aestivum-H. villosa chromosome addition, substitution and translocation lines involved in different H. villosa accessions and wheat backgrounds. Therefore, these chromosome-specific microsatellite markers could be used as molecular markers for detection of chromosomes of H. villosa in common wheat.